Transgenic mouse expressing the human cyclooxygenase-2 gene and neuronal cell cultures derived therefrom

ABSTRACT

The invention provides for transgenic mice whose genomes comprise a gene encoding human cyclooxygenase-2 under the control of a neuron-specific promoter. The transgenic mice of the instant invention express human cyclooxygenase-2 mRNA in neuronal cells of their hippocampi at increased levels relative to the level of human cyclooxygenase-2 mRNA expressed in their cerebral white matter. Moreover, neuronal cell cultures derived from the transgenic mice of the instant invention arc more susceptible to aggregated Aβ25-35 peptide-mediated impairment of redox activity relative to those derived from mice non-transgenic for the human cyclooxygenase-2 gene. These transgenic animals and the neuronal cultures derived therefrom are useful iii elucidating the pathophysiological bases of neurodegenerative diseases and in improving the diagnosis and treatment of these disorders.

This application is a national stage of International Application No.PCT/US97/21484, is a continuation of U.S. patent application Ser. No.08/831,402, filed Apr. 1, 1997, now U.S. Pat. No. 5,985,930, andprovisional application No. 60/033,332, filed Nov. 21, 1996.

1. INTRODUCTION

The present invention relates to the use of nimesulide and structurallyrelated compounds in the prevention and/or treatment ofneurodegenerative conditions such as Alzheimer's Disease. It is based,at least in part, on the discovery that nimesulide, in effectiveconcentrations, inhibits cell death.

2. BACKGROUND OF THE INVENTION 2.1. Alzheimer's Disease

Sporadic Alzheimer's Disease is the major neurodegenerative diseaseassociated with aging, the risk of developing the disease risingexponentially between the ages of 65 and 85, doubling every five years.Histologically, the hallmarks of Alzheimer's Disease are the depositionof amyloid in senile plaques and in the walls of cerebral blood vessels;the presence of neurofibrillary tangles, and neurodegeneration. Theetiology of Alzheimer's Disease, however, is not well understood.Genetic factors have been proposed to play a role, including trisomy 21,mutations in the amyloid β-protein precursor (“APP”) gene, thepresenilin-1 (PS 1) and presenilin-2 (PS2) genes, and the presence ofthe apolipoprotein E type 4 allele (Younkin, 1995, Ann. Neurol.37:287-288; Lendon et al., 1997, JAM A 277:825). Several studies haveindicated that β-amyloid induces apoptosis in cultured neurons (Loo etal., 1993, Proc. Natl. Acad. Sci. U.S.A. 90:7951-7955; Li et al., 1996,Brain Res. 738:196-204). Such induction may involve the immediate earlygene proteins, c-jun and fos (Anderson et al., 1995, J. Neurochemistry65:1487-1498; Anderson et al., 1994, Experimental Neurology 125:286-295;Anderson et al., 1996, J. Neurosci. 16:1710-1719).

It has been proposed that apoptosis may be involved in the pathogenesisof Alzheimer's disease (Smale et al., 1995, Exp. Neurol. 133:225-230;Anderson et al., 1996, J. Neurosci. 16:1710-1719; Anderson et al., 1994,Exp. Neurol. 125:286-295). Inflammatory mechanisms have also beenimplicated; (Pasinetti, 1996, Neurobiol. Ageing 17:707-716) supportiveof such a mechanism are the observations that acute phase proteins areelevated in the serum of Alzheimer's Disease patients, and are depositedin amyloid plaques; activated microglial cells tend to localize in thevicinity of senile plaques, and complement components have beenlocalized around dystrophic neurites and neurofibrillary tangles (Aisenand Davis, 1994, Am.J.Psychiatry 151:1105-1113). Antiinflammatory agentshave been suggested as potential therapeutic agents (Aisen et al., 1996,Dementia 7:201-206; McGeer et al., 1996, Neurology 47: 425-432). Becausethey tend to have fewer adverse side effects, selective inhibitors ofthe enzyme cyclooxygenase-2 have been advanced as agents for treatingsuch inflammation (International Publication No. WO 94/13635 by MerckFrosst Canada Inc.). Prior to the present invention, however, it had notbeen believed that such agents could be used to inhibit non-inflammatoryaspects of neurodegeneration in the context of Alzheimer's Disease orotherwise.

2.2. Cyclooxygenase-2

Cyclooxygenases (“COXs”) are enzymes that catalyze the formation ofprostaglandin (“PG”)-H₂ from arachidonic acid (AA). PG-H₂ is furthermetabolized to physiologically active PGs (e.g., PG-D₂, PG-E₂ andPG-F_(2α)), prostacyclin (PG-I_(2α)) and thromboxanes. Specific PGs havediverse, often antagonistic effects on different tissues. For example,PG-I₂ and PG-E₂ are potent vasodilators that may contribute to theinflammatory response, whereas PG-F_(2α), is a vasoconstrictor.

There are two known COX isoforms, COX-1 and COX-2, which, thoughphysiologically distinct, are similar in amino acid sequence andenzymatic functions. COX-1 is constitutively expressed at differentlevels in different cell types. COX-2, however, is not constitutivelyexpressed, and is generally undetectable in normal peripheral tissues(Kujubu et al., 1991, J. Biol. Chem. 266:12866-12872; O'Banion et al.1992, Proc. Natl. Acad. Sci. U.S.A. 89:4888-4892). Rather, COX-2expression is inducible (for example, by mitogens) and COX-2 mRNA levelshave been observed to rise rapidly in response to inflammatory stimulisuch as interleukin-1β and lipopolysaccharide, and to decrease inresponse to glucocorticoids. When subjected to these same factors, COX-1mRNA levels remain substantially unchanged, suggesting that COX-2 is theisoform which mediates inflammation (Cao et al., 1995, Brain Res.697:187-196; O'Banion et al. 1992, Proc. Natl. Acad. Sci. U.S.A.89:4888-4892).

Recent evidence suggests that COX-2 may play a role in mechanisms ofcell survival and cell adhesion in peripheral cells (Lu et al., 1996,Proc. Natl. Acad. Sci. U.S.A. 92:7961-7965; Tsujii et al., 1995, Cell83:493-501). Tsujii et al. reports that epithelial cells engineered toexpress elevated levels of COX-2 were resistant to butyrate-inducedapoptosis, exhibited elevated BCL2 protein expression, and reducedtransforming growth factor β2 receptor levels (Tsuji et al., 1995, Cell83:493-501). Lu et al. indicates that non-steroidal antiinflammatorydrugs may induce an apoptotic mechanism involving the COX system.

The roles of COX-1, COX-2 and PG synthesis in normal brain, and in thecontext of Alzheimer's Disease, have not been fully characterized todate. The importance of PGs in brain physiology may be independent ofinflammatory mechanisms. In the brain, PG receptors have been identifiedin the hypothalamus, thalamus, and limbic system (Watanabe et al., 1989,Brain Res. 478:143-148). PGs are involved in hypothalamic-pituitaryhormone secretion (Kinoshita et al., 1982, Endocrinol. 110:2207-2209),regulation of temperature and the sleep-wake cycle (Hayaishi, 1988, J.Biol. Chem. 263:14593-14596). There is recent evidence that COX-2 mRNAis expressed and regulated in rat brain by synaptic activity andglucocorticoids (Adams et al., 1996, J. Neurochem. 66:6-13; Kaufmann etal., Proc. Natl. Acad. Sci. U.S.A. 93:2317-2321; Yamagata et al., 1993,Neuron 11:371-386). These studies indicate that COX-2 is regulated as animmediate early gene in the brain, and suggest that PGs may be importantin trans-synaptic signaling and long-term potentiation. Chang et al.(1996, Neurobiol. of Aging 17:801-808) report that COX-2 mRNA expressionis decreased in Alzheimer's disease.

3. SUMMARY OF THE INVENTION

The present invention relates to the use of nimesulide and structurallyrelated compounds in the prevention and/or treatment ofneurodegenerative conditions. It is based, at least in part, on thediscoveries that (i) COX-2 expression in models of neurodegeneration isincreased in neurons rather than glial cells (consistent with anon-inflammatory mechanism), and (ii) nimesulide exhibits aneuroprotective effect against β-amyloid induced neuronal cell death.This latter finding is particularly unexpected in view of the ability ofCOX-inhibitors to increase apoptosis of non-neuronal cells. Withoutbeing bound to any particular theory, it appears that nimesulideinhibits a non-inflammatory mechanism of neurodegeneration.

4. DESCRIPTION OF THE FIGURES

FIG. 1. Regional expression of COX-2 mRNA in control unlesioned male ratbrain. COX-2 mRNA expression was assessed by in situ hybridization andvisualized by X-ray autoradiography. Abbreviations: DG, dentate gyrus;CA1, CA2 and CA3 subregions of the neuronal pyramidal layer of thehippocampal formation; PAC, parietal cortex; PYC, pyriform cortex; AC,amygdaloid complex; THAL, ventroposterior thalamic nucleic. Adapted fromplate 24 of Paxinos and Watson, 1986 (The Rat Brain in SteriotaxicCoordinates, Academic Press, NY).

FIG. 2. Maturational regulation of COX-2 mRNA expression in rat brain.Optical densities were quantified from autoradiographic images.Abbreviations, as above and as follows: PYC/AC, pyriform and amygdaloidcomplex that were quantified for COX-2 mRNA expression as a single brainregion. N=5-8 per time point.

FIG. 3. Maturational influence on COX-2 mRNA expression during responseto KA-induced seizures. Micrographs were generated from autoradiographicimages. For anatomical distribution of changes, refer to FIG. 1.Control, unlesioned vehicle-injected rats; KA 8 H, KA-treated rats 8hours prior to sacrifice; postnatal days P-7, P-14, and P-21.

FIGS. 4A-D. Time course of COX-2 mRNA changes in rat brain duringresponses to KA treatment: maturational influences and regionaldistribution of changes. Optical densities were quantified fromautoradiographic images. Data are expressed as means±SEM, N=4=6 pergroup. *P<0.01 vs 0 H group (saline injected group); 4 h, 8 h, 16 h, 30h, 120 h, time in hours after onset of KA-induced seizures.

FIGS. 5A-D. Maturational influence on the distribution of COX-2 mRNAexpression and induction in rat hippocampus. COX-2 mRNA in P21 (A,B) andadult (C,D) hippocampal formations as assessed by in situ hybridizationassay and visualized by emulsion autoradiography using dark-fieldillumination. In A and C, COX-2 mRNA expression in control rats (vehicleinjected); in B and D, COX-2 mRNA 8 hours after onset of KA-inducedseizures. Arrows point toward the superficial layer of the DG (stratumgranulosum). Scale bar=200 μm.

FIG. 6. Selective induction of hippocampal COX-2 but not COX-1 mRNAsduring response to KA-induced seizures as assessed by gel blothybridization assay. CTL, control saline-injected rats; KA, KA-treatedrats 12 hours post lesioning.

FIGS. 7A-I. KA-induced COX-2 and apoptosis in adult rat brain. In (A,B),COX-2 mRNA expression in CA3 hippocampal pyramidal neurons of controland KA-treated rat, respectively; in (C), CA3 subregion of thehippocampal formation of KA-lesioned rats showing neurons with apoptoticfeatures (arrows). In (D,E), COX-2 mRNA expression in pyriform cortex ofcontrol and KA-treated rat respectively. In (F), arrows point towardapoptotic cells of pyriform cortex of KA-lesioned rats. In (G,H), COX-2mRNA expression in cells of the amygdaloid complex of control andKA-treated rats, respectively. In (I), arrows point toward apoptoticcells of the amygdaloid complex of KA-lesioned rats. COX-2 mRNA wasassayed by in situ hybridization and visualized by emulsionautoradiography under dark field illumination. In situ 3′ end-labelingwas used to assess apoptotic features following KA treatment. Scale bar:in A,B,D,E,G,and H=200 μm; in C,F and I=40 μm.

FIGS. 8A-D. Immunocytochemical evidence of neuronal COX-2expression/regulation in response to glutamate in vitro. COX-2-likeimmunoreactivity in monotypic cultures of rat primary hippocampalneurons in (A) control and (B) after glutamate exposure (12 hours). In(C,D) control and glutamate treated cultures immunoreacted withimmunoadsorbed COX-2 antibody, respectively. Scale bar=50 μm.

FIGS. 9A-C. Effect of nimesulide on endotoxin-mediatedsynthesis/secretion of cytokines and nitrites in glia. The effect of10⁻⁹ M nimesulide on the synthesis/secretion of (A) TNF, (B) NOintermediates (Griess reaction) and (C) PGE₂, as assessed in BV2 mouseimmortalized microglial cells. Mean±SEM, n=8-10 per group.Lipopolysaccharide (“LPS”)=5 μg/ml. LPS and nimesulide were added incombination to cultures; incubation time was 24 hours. Statistics usedANOVA, p<0.05.

FIGS. 10A-E. Time course of changes of COX-2 protein in P19 cells duringresponse to conditions leading to apoptotic death. In (A), quantitativeanalysis of COX-2 induction in P19 cells, n=4-6 per group, p<0.05 vs.t=0. In (B), changes were assessed by western analysis, usingchemiluminescent detection. In (C,D) the morphological appearance ofapoptotic nuclei in P19 cells was assessed by Hoechst H33258 24 hoursafter serum removal (and replacement with N2 medium). In (E), theelectrophoretic profile of DNA showing DNA laddering degradation wasassessed 14 hours after serum removal (lane 1, control cells at t=0;lane 2, DNA laddering 14 hours after serum removal; lane 3, DNAmarkers). The COX-2 polyclonal antibody used in these studies was asdescribed in Section 6. The COX-2 specific antibody recognizes two majorbands having estimated molecular weights of about 70 and 65 kDa in totalhomogenates of mouse, rat and human brains.

FIGS. 11A-E. Regulation of COX-2 during response to Aβ1-40 mediatedoxidative stress in SH-SY5Y neuronal cells. (A) Bar graph showing theamount of COX-2 immunoreactivity present relative to control (“CTL”).The immunoreactivity measurement was derived from the data shown in (B).(B) Western blot of proteins prepared from control SH-SY5Y cells orcells exposed to Aβ peptides for 48 or 72 hours, reacted with COX-2antisera. (C) Results of MTT assays of control or Aβ-treated SH-SY5Ycells. (D) The immunoblot shown in (B) stripped and immunoreacted withactin antisera. (E) SH-SY5Y cultures were examined for apoptoticmechanisms.

FIG. 12. Protective effect of nimesulide on Aβ1-40 mediatedneurotoxicity as assessed by MTT assay using SH-SY5Y neuronal cells.Abbreviations: CTL control; NIM nimesulide; A B-β-amyloid (1-40).*P<0.01 vs CTL, **P<0.05 vs. CTL.

FIG. 13. Micrographs showing induction of COX-2 immunoreactivity intemporal cortex of AD and age-matched neurological controls. Themicrographs show a selective induction of COX-2 immunostaining inneuron-rich grey matter (“gm”) but not glia-rich white matter (“wm”)regions.

FIGS. 14A-D. COX-2 immunostaining of neurons in frontal cortex of ADbrain and co-localization of COX-2 immunostaining with AD plaques infrontal cortex of AD brains. In (A,B), immunostaining of COX-2 neuronsof AD are shown (A, low power; B, high power magnification). In (C),immunostaining of diffuse plaques. In (D), COX-2 in neuritic plaques asassessed by Aβ immunostaining on adjacent tissue sections.

FIGS. 15A-E. COX-2 expression in AD brain frontal cortex. (A) Northernblots of COX-2 and COX-1 mRNA in AD frontal cortex relative to controlsquantitative analysis of data is in (B). (C) Western blots of COX-2protein in AD frontal cortex and control; quantitative analysis of datais in (D).

FIG. 16. COX-2 mRNA regulation in neurons of the anterior and posteriorhorn of the spinal cord of ALS and neurological controls.Autoradiographic images were visualized by X-ray film. The arrow pointedtoward the ventral horn shows intense induction of COX-2 mRNA signal inthe ALS case.

FIGS. 17A-D. COX-2 mRNA elevation in a biopsy of human corticalepileptic foci as assessed by in situ hybridization assay.Autoradiographic images were visualized by X-ray film. Arrow pointedtoward the cortical layers showing intense COX-2 mRNA signal inepileptic brain (A) versus control (B). Parts (C) and (D) show analysisof compiled data relating to COX-2 and COX-1 mRNA levels.

FIG. 18. Potentiation of cyclooxygenase and peroxidase activities byaggregated Aβ peptides.

FIGS. 19A-B. Transient expression of COX-2 in neuronal cells potentiatesAβ-mediated impairment of redox activity. SH-SY5Y neuronal cells weretransfected with either the human COX-2 (A) or COX-1 (B) gene andtreated with Aβ₂₅₋₃₅, and then redox activity was assessed by MTT assay.

FIGS. 20A-B. Generation of Transgenic Mice Expressing COX-2. (A) In situhybridization demonstrates COX-2 mRNA expression in TgNHC32 mice but notwild-type (mice one month old). (B) Regional expression of hCOX-2 mRNAin NHC32 and NHC5 relative to wild-type, as quantified using Bioquantimage-analysis.

FIGS. 21A-C. hCOX-2 over-expression potentiates Aβ-mediated oxidativeimpairment in primary mouse neuronal cultures.

FIGS. 22A-C. Nimesulide protects B12 neuronal cells against glutamatemediated oxidative stress.

FIGS. 23A-C. Control studies for FIG. 22. Dose (A) and time course (B)association with glutamate mediated oxidative impairment as measured byLDH assay. (C) demonstrates lack of toxicity of nimesulide to B12cultures.

5. DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to the use of the cyclooxygenase-2(“COX-2”) selective inhibitor, nimesulide, and structurally relatedcompounds in the prevention and/or treatment of neurodegenerativeconditions. It is based, at least in part, on the discoveries that COX-2expression increased in parallel with neuronal lesions produced bykainic acid and with induction of neuronal apoptosis in an establishedin vitro system (see Sections 6 and 8, below). Contrary to reports inthe prior art that COX-2 plays a primary role in inflammation and may beinvolved in an inflammatory component of neurodegeneration (for example,in the context of Alzheimer's Disease), it has further been discoveredthat in the human brain, COX-2 expression appears to be restricted toneurons rather than to glial cells (whereas expression in glial cellswould be expected if an inflammatory mechanism were operative: seeSection 9, below). A correlation between COX-2 expression and thecharacteristic pathological change associated with Alzheimer's Disease,amyloid plaque, has been observed (see Section 9). Further, it has beendemonstrated that nimesulide exerts a neuroprotective effect againstβ-amyloid induced cell death in neuronal cultures. In view of thesefindings, according to the invention, nimesulide and related compoundsmay be used to intervene in the process of apoptotic neuronal cell deathassociated with Alzheimer's Disease and other neurodegenerativeconditions. Without being bound to any particular theory, the action ofnimesulide and related compounds may be mediated by the reduction of theapoptotic effects of oxidative stress.

The term “nimesulide”, as used herein, refers to a compound,4-nitro-2-phenoxymethanesulfonaninide, having a structure as set forthfor Formula I below.

Compounds which are considered structurally related are compounds whichhave a similar bicyclic structure and which selectively inhibit COX-2.For example, substituents, analogs, and enantiomers of nimesulide areconsidered to be structurally related compounds. As a further example, astructurally related compound may compete with nimesulide for binding toCOX-2, or may bind to substantially the same location of COX-2, asdetermined crystallographically. Moreover, nimesulide, conjugated toanother compound, is also considered to be a structurally relatedcompound as defined herein.

Nimesulide, or a structurally related compound, may be administered soas to provide an effective concentration in the nervous system of thesubject being treated. An effective concentration is defined herein asthat concentration which inhibits neuronal cell death by at least 20percent. In specific, nonlimiting embodiments of the invention, theconcentration of nimesulide is at least 1 nanomolar, and preferably atleast 1 micromolar in the location of neuronal cells which are desiredto be treated. Desirably, the concentration of nimesulide is less than10⁻³-molar to avoid toxicity. In one such specific embodiment, where theneurodegenerative condition to be treated is Alzheimer's Disease, theconcentration of nimesulide in the hippocampal formation of the subjectis at least 1 picomolar, preferably at least 1 nanomolar, and morepreferably at least 1 micromolar. The corresponding serum concentrationsmay be at least 10 picomolar, preferably at least 10 nanomolar, and morepreferably at least 10 micromolar. Equivalent amounts of structurallyrelated compounds (adjusted to compensate for differences in potency)may also be used.

Nimesulide or a structurally related compound may be administered in anymanner which achieves the desired effective concentration. For example,suitable routes of administration include oral, intravenous,subcutaneous, intramuscular, transdermal and intrathecal routes.

Nimesulide or a structurally related compound may be comprised in asuitable pharmaceutic carrier. Formulations may provide for sustainedrelease.

For example, but not by way of limitation, nimesulide may beadministered orally at doses of 2-800 mg/day, preferably 50-400 mg/day,and most preferably 200 mg/day.

Neurodegenerative conditions which may be treated according to theinvention include, but are not limited to, Alzheimer's Disease,Parkinson's Disease, seizure-associated neurodegeneration, amyotrophiclateral sclerosis, spinal cord injury and other diseases wherein thecondition is not conventionally regarded as an inflammation-mediated orautoimmune disorder.

6. EXAMPLE Maturational Regulation and Regional Induction ofCyclooxygenase-2 in rat Brain

6.1. Materials and Methods

Animals and excitotoxic lesions. Male adult Sprague/Dawley rats ofdifferent ages were maintained in a controlled light and temperatureenvironment, with food and water ad libidum. In adult rats (250-300 g),hippocampal excitotoxic lesions were induced by subcutaneous injectionof kainic acid (“KA”; 10 mg/kg, Sigma). Because KA uptake is higher inyoung rats relative to adults (Berger et al., 1986, in Schwartz andBen-Ari, Advances in Experimental Medicine and Biology, Plenum, N.Y.,pp. 199-209), KA doses were adjusted to produce maximal excitotoxicitywithout reaching lethal doses (from 2 mg/kg at postnatal day P-7 to 6mg/kg at postnatal day P25). Saline injected rats were used as controls(0 hour time point).

COX-1 and COX-2 cDNA probes. Bluescript plasmid (Stratagene) containingthe full length rat COX-1 cDNA (2.7 kb) was linearized by digestion withClaI; PCRII plasmid (Invitrogen) containing the coding sequence for ratCOX-2 (1.8 kb) was linearized by digestion with PflMI (Feng et al.,1993,Arch. Biochem.

Biophys. 307:361-368). Linearized plasmids were purified using Elu-Quick(Schleicher & Schuell) after agarose gel electrophoresis.

In situ hybridization. At various intervals after the onset ofKA-induced seizures, the rats were sacrificed, and the brains quicklyremoved, rinsed in cold phosphate buffer (PBS, 10 mM, pH 7.4) andimmersed in methylbutane at −25° C. for three minutes. The brains weresliced into 10 micrometer sections, frozen, and the resulting frozensections were mounted on polylysine-coated slides and stored at −70° C.For immunocytochemistry (“ICC”) or in situ hybridization (“ISH”), frozensections were post-fixed in PBS containing 4 percent paraformaldehyde(30 minutes at room temperature) and then rinsed in PBS. For ISH, tissuesections were rinsed in 0.1M triethanolamine (“TEA”), pH 8.0, incubatedin acetic anhydride (“AAH”; 0.25% v/v in TEA, 10 minutes) and rinsed inTEA and PBS. Following AAH treatment, tissue sections were hybridizedwith [³⁵S]-cRNA probes (0.3 μg/ml, 2×10⁹ dpm μg-⁻¹) made from COX-2linearized cDNA transcription vectors (Feng et al., 1993, Arch. Biochem.Biophys. 307:361-368). Sense strand hybridization was used as a controland gave negative results. Following hybridization for 3 hours at 50°C., stringent washes (0.1×SSC at 60° C.) and dehydration, slides wereexposed to X-ray film for seven days for quantification. Slides werethen exposed to NTB-2 emulsion (Kodak, Rochester, N.Y.) for microscopicanalysis of COX-2 mRNA distribution. Following development, tissuesections were counterstained with cresyl violet. Film autoradiogramswere analyzed quantitatively using an image analysis system withsoftware from Drexel University (Tocco et al., 1992, Eur. J. Neurosci.4:1093-1103). Statistics were calculated by ANOVA followed by additionalposthoc analysis.

In situ end labeling. In parallel studies, paraformaldehyde-fixed braintissue sections were dehydrated, air dried and incubated with dATP,dCTP, dGTP (0.2 mM), dTTP (13 μM), digoxigenin-11-dUTP and DNApolymerase I (Boehringer Mannheim) at 10 units/100 μl at 37° C. for 2hours. The reaction was stopped by addition of 20 mM EDTA, pH 8.0.Sections were incubated at room temperature overnight with analkaline-phosphatase-conjugated digoxigenin antibody (Genius System,Boehringer Mannheim) diluted 1:200 in 5 percent sheep serum diluted in150 mM NaCl, 100 mM TRIS-HCl, pH 7.5. Colorimetric detection with nitroblue tetrazolium and 5-bromo-4-chloro-3-indolyl-phosphate was performedwith the Genius System by the manufacturer's protocol (Sakhi et al.,Proc. Natl. Acad. Sci. U.S.A. 91:7525-7529).

Northern blot hybridization assay. RNA extraction was performed asfollows. After sacrifice, rat brains were dissected and stored at −75°C. prior to processing. Total RNA was extracted from pools ofhippocampal tissue (Pasinetti et al.,1994, J. Comp. Neurol.339:387-400). Briefly, tissues were homogenized for 1 minute in 4 Mguanidinium thiocyanate, 25 mM sodium citrate (pH 7.5), 0.5% sarcosyland 0.1 M β-mercaptoethanol, in a final volume of 0.5 ml. Afteracidified phenol/chloroform extraction and ethanol precipitation, theRNA pellet was rinsed consecutively with 70% and 100% ethanol. Thepurified RNA was then dissolved in 0.5% sodium dodecyl sulfate (“SDS”)and stored at −75° C. Total RNA was quantified in a UVspectrophotometer. Total RNA (5 μg) from tissue was electrophoresed ondenaturing (0.2M formaldehyde) agarose gels and transferred to a nylonmembrane (Nylon 66 plus; Hoeffer, San Francisco Calif.) in 2×SSC. Blothybridization was carried out with 10⁶ cpm/ml of antisense COX-1 orCOX-2 [³²P]-cRNA probes in 50% formamide, 1.5×SSPE, 1% SDS, 0.5% drymilk, 100 μg/ml yeast total RNA and 500 mg/ml salmon sperm DNA at 53° C.for about 15 hours. Blots were washed to a final stringency of 0.2×SSC,0.2% SDS at 72° C. Blots were exposed to Kodak X-ray film (XAR-5) withintensifying screens at −70° C.

Cell cultures. Hippocampal neuron cultures were derived from embryonicrat brain. E16-E18 embryos were dissected in Hank's balanced saltsolution and cultured (Pasinetti et al.,1994, J. Comp. Neurol.339:387-400; Peterson et al., 1989, Dev. Brain Res. 48:187-195). Culturemedia were changed every 3 days. For glutamate neurotoxicity studies,eight day old cultures were treated with glutamate (250 μM, Sigma) inthe presence of 2.4 mM calcium ion and 0.8 mM magnesium ion. After 6hours of glutamate exposure, culture medium was replaced with freshmedium; COX-2-like immunoreactivity was assessed in neurons 12 hourslater.

Immunocytochemical detection of COX-2 in monotypic cultures of primaryrat neurons. Control and glutamate treated cultures were post-fixed inPBS containing 4% paraformaldehyde (30 minutes, room temperature),rinsed in PBS, pre-treated with normal serum and incubated overnight at4° C. with primary antibodies. COX-2 antisera (rabbit IgG) was raisedagainst a synthetic peptide (CNASASHSRLDDINPT; SEQ ID NO:1) encompassingthe C-terminal region of the murine COX-2. The antisera reacts withhuman and rat COX-2 but not with COX-1, as assessed by Western blotanalysis. Vectastain ABC kit (Vector, Burlingame, Calif.) was used insubsequent steps to complete the diaminobenzene staining (Pasinetti etal.,1994, J. Comp. Neurol. 339:387-400). Immunoadsorption of COX-2antisera with synthetic COX-2 peptides controlled for specificity;adsorption was carried out overnight at 4° C. with synthetic COX-2peptides at 30 μg/ml.

6.2. Results

COX-2 expression in adult rat brain. FIG. 1 shows by ISH that theregional distribution of COX-2 mRNA was most notable in limbicstructures, but was also present in neocortex (consistent with thereports of Kaufmann et al., Proc. Natl. Acad. Sci. U.S.A. 93:2317-2321and Yamagata et al., 1993, Neuron 11:371-386). In the hippocampalformation, COX-2 mRNA was selectively expressed in cells of the granuleand pyramidal neuron layers. COX-2 mRNA expression was also found in theouter layer of the parietal cortex, the pyriform cortex and cells of theamygdaloid complex.

Maturational regulation of hippocampal COX-2 mRNA expression. ISHresults indicated that during maturation, COX-2 mRNA expression isdifferentially regulated in subsets of cells of neuronal layers of thehippocampal formation (FIG. 2, FIG. 3 top). From postnatal days P7-P14,COX-2 mRNA showed greater than two-fold increase in the granule celllayer of the dentate gyrus and in the CA3 subdivision of the pyramidalcell layer (p<0.001, FIG. 2). Though the expression of COX-2 mRNA waslower in the other brain regions examined, the pattern of maturationalexpression was similar (FIG. 2). By postnatal day P21, COX-2 mRNAexpression approximated adult levels in all subregions examined (FIG. 2,FIG. 3 top).

Response to KA-induced seizures. To further explore maturationalregulation of COX-2 in brain, COX-2 mRNA expression during responses toKA-induced seizures was examined postnatally. Despite intense seizureactivity after KA treatment, no detectable change of COX-2 mRNAexpression was found in any brain region examined at P7 (FIG. 3, FIG.4A). Changes in COX-2 mRNA expression at 120 hours post-KA treatment inthe P7 group indicate developmental maturation rather than response toKA toxicity (FIG. 4A). In contrast to the P7 group, at P14 and P21 COX-2mRNA increased within 4-8 hours after onset of KA-induced seizures inall the hippocampal subregions examined (FIGS. 4B and 4C). The level ofCOX-2 mRNA expression returned toward control levels within 120 hoursafter treatment in P14, P21 and adult rat brain.

Within the dentate gyrus, control COX-2 expression in P14 and P21 ratswas asymmetric, selectively localized to the more superficial neurons ofthe stratum granulosum rather than the deeper granule cells of thedentate gyrus blade (FIG. 5A). In response to KA treatment, COX-2 mRNAinduction showed similar asymmetry of expression (FIG. 5B). In contrast,the asymmetry within the dentate gyrus was less notable in controlanimals and after KA-induction of the adult group (FIGS. 5C and 5D).

In parallel studies, northern blot hybridization of total RNA fromhippocampus of adult rats 12 hours after KA-induced seizures confirmedCOX-2 mRNA induction (FIG. 6). No detectable induction of COX-1 mRNA wasfound in the same rat brain (FIG. 6).

KA-induced COX-2 and apoptosis in adult rat. By 8 hours after onset ofKA-induced seizures, COX-2 mRNA induction in cells of the CA3 region ofthe hippocampal formation (FIG. 7B), pyriform cortex (FIG. 7E) andamygdaloid complex (FIG. 7H) of the adult brain paralleled temporallyand overlapped anatomically the onset of apoptosis as assessed by insitu end-labeling in the same brain regions (FIG. 7C, CA3 regions of thehippocampus; FIG. 7F, pyriform cortex; FIG. 7I, amygdaloid complex).Cellular COX-2 mRNA expression in adult, control (FIGS. 7A, 7D and 7G)and KA-treated (FIGS. 7B, 7E and 7H) rats was identified by emulsionautoradiography using ISH assays.

Immunocytochemical evidence of neuronal COX-2 expression/regulation inresponse to glutamate in vitro. Primary cultures of rat hippocampalneurons were exposed to glutamate in vitro. At baseline, constitutiveCOX-2 expression was demonstrated by immunocytochemistry (FIG. 8B).Twelve hours after exposure to glutamate, an increase in COX-2immunoreactivity was observed which coincided with marked reduction inthe number of neurons (FIG. 8D).

COX-2 expression in human epilepsy. Increased expression of COX-2 mRNAwas detected by ISH in a biopsy of human brain at epileptic foci (FIGS.17A-D). It was found that COX-2 mRNA, but not COX-1 mRNA, wassignificantly elevated in the cortex of epileptic patients the p vlauewas <0.05.

7. EXAMPLE Nimesulide Suppresses Cytokine and Nitrite Production inMicroglia Cultures

In experiments conducted in vitro, nimesulide at low concentration (1nanomolar) was found to be effective in the suppression ofendotoxin-mediated induction of tumor necrosis factor (“TNF”) productionby immortalized brain-derived microglia (BV-2) and astrocytes (FIG. 9A).In parallel, nimesulide was equally effective in blocking nitriteproduction (Griess reaction; FIG. 9B). This latter observation isparticularly relevant in view of evidence showing that blockade ofneuronal nitric oxide (“NO”)-synthase protects against glutamateneurotoxicity. Nimesulide was also found to be effective in blockingendotoxin-mediated induction of prostaglandin PGE₂) in brain-derivedmicroglia (FIG. 9C).

8. EXAMPLE COX-2 Expression in Apoptotic Cells

The regulation of COX-2 expression was studied using an established invitro model of apoptosis. Specifically, the regulation of COX-2 wasstudied in P19 embryonic carcinoma cells during responses to serumdeprivation. Under such conditions, the P19 cells underwent apoptoticcell death, showing characteristic DNA fragmentation and nuclearmorphology. As shown in FIG. 10, using this system, coincidental onsetof apoptosis and elevation of COX-2 expression was observed.

These studies were extended to the human neuronal cell line SH-SY5Y.β-amyloid has been demonstrated to play a role in inducingneurodegeneration in SH-SY5Y cells (Oda et al., 1995, Alzheimers Res.1:29-34). SH-SY5Y cells were treated with synthetic aggregated Aβ₁₋₄₀peptides at a concentration of 20 μM for various periods of time.Western blot analysis was performed of cell extracts, using COX-2antisera or actin antisera as control (FIGS. 11B and D, respectively).In FIG. 11A, COX-2 protein from control or Aβ-treated SH-Sy5Y neuronalcells was quantified from the immunoblots shown in FIG. 11B (at the 72hr. time point; only the 70 kDa mw species being quantified). COX-2signal was abolished by immunoadsorption of COX-2 antisera with purifiedhuman recombinant COX-2 (“hrCOX-2”) peptides. FIG. 11C shows thatdiminished cell redox activity was observed 72 hours after treatmentwith aggregated Aβ₁₋₄₀ (at 20 μm), as assessed by MTT assay in parallelcultures. Bar graphs represent the mean±SEM, n=4-5 per group, p<0.05(t-test). COX-2 integrated optical densities were analyzed fromdigitized images using a Bioquant image analysis (Biometrics, Nashville,Tenn.). It was also found that COX-2 expression occurred in parallel toDNA laddering (FIG. 11E), an index of apoptosis. FIG. 12 shows thatnimesulide at 10⁻⁶ and 10⁻⁹ molar was able to block Aβ1-40 toxicity, asassessed by the MTT assay.

Interestingly, when aggregated synthetic Aβ₁₋₄₀ peptides (150 μM) werecoincubated with purified hr-holoCOX-2 (COX-2 plus heme-cofactor, 50 nM)for 16 hours, at 37° C. in 1X PBS, and cyclooxygenase and peroxidaseactivities were measured (Murphy et al., 1989, Neuron 2:1547-1558)cyclooxygenase and peroxidase activities increased (relative to activitylevels of enzyme in the absence of Aβ; FIG. 18). Cyclooxygenase andperoxidase levels in the absence of heme gave negative results.

9. EXAMPLE COX-2 Expression in Alzheimer's Disease

The expression of COX-2 in normal human brains and in brains ofAlzheimer's Disease (“AD”) patients was studied. Immunocytochemical dataindicates that COX-2 expression is primarily localized to cells withneuronal morphology in human brain; minimal localization of COX-2immunostaining in cells with glial morphology was found in any regionexamined. These results tend to suggest a role for COX-2 in anon-inflammatory function. While the immunocytochemical signal for COX-2was at the limit of detection in temporal cortex of neurological controlcases, COX-2 showed elevation in AD brain (FIGS. 13A, 13B) The selectiveinduction of COX-2 immunoreactivity in grey matter is consistent withthe findings showing neuronal induction of COX-2 in rat brain duringresponses to lesions leading to neuronal death (see Section 6).

The cellular immunodistribution of COX-2 in AD brain was also explored.It was observed that neuronal COX-2 is not only localized to theperikarya (FIG. 14A, but is found in neuronal projections as well (FIG.14B). Moreover, intense immunostaining was also found in diffuse plaques(FIG. 14C) and in AD neuritic plaques identified by Aβ immunostaining onadjacent tissue sections of the hippocampal formation. These findingssuggest a role for COX-2 in mechanisms of neuronal death or survival.

Studies were performed to quantify COX-2 expression in brains of AD andage-matched controls. We used quantitative dot-blot analysis andchemiluminescence detection. Western analysis was used for qualitativeassessment. A greater than 2-fold elevation of COX-2 content was foundin hippocampal homogenates of AD brains compared to neurologicalage-matched controls.

Because of the immunocytochemical evidence showing COX-2immunoreactivity in AD plaques, levels of COX-2 and total Aβ in AD caseswere compared. A direct correlation was found.

COX-2 mRNA and protein were found to be increased in AD brain frontalcortex. FIG. 15A shows Northern blots of total RNA from neurologicalcontrol (C) and AD frontal cortex hybridized to [32p] COX-2 and COX-1mRNAs. The mRNA species detected by COX-2 and COX-1 cRNA probesrepresent an RNA of the same size found in peripheral tissues. FIG. 15Bshows quantified results of the Northern studies. Specifically, COX-2mRNA prevalence was increased in the frontal cortex of AD vs control,where the presence of equal amounts of RNA in both lanes was confirmedby normalization to 28S rRNA in the hybridization membrane, and completetransfer of RNA to the membrane was evidenced by the absence of 28S and18S rRNAs in the gel post transfer. Integrated optical densities wereanalyzed from digitized images using a Biquant image analysis. Bargraphs represent n=9 per group, and the results were significant by at-test value of p<0.05.

Similarly, when Western blot analysis was performed of AD frontal cortexvs. controls, the amount of COX-2 protein was found to increase. FIG.15C shows the results of such a western blot. Lanes 1 and 2 show theCOX-2 protein content of the same tissues uses for Northern analysis.Lanes 3-4 show the absence of COX-2 bound when COX-2 antisera wasimmunoadsorbed using hrCOX-2 peptides, demonstrating the specificity ofthe signal.

The expected 70 kDa new species of hrCOX-2 (lane 5) also was absent whenantisera was preadsorbed with hr COX-2 peptides. These results arequantified in FIG. 15D (with respect to the 70 kDa species). Further,the immunoblots were stripped and imnnunoreacted with actin antisera toverify the specificity of changes (actin values: control group 100±8; AD94±7% of control Bar graphs represent n=9 per group; t-test value isp<0.05). The inset in FIG. 15D shows a correlation of COX-2 content andthe number of plaques per mm², n=8, r=0.72, p<0.03. COX-2 and actinintegrated optical densities were analyzed from digititzed images usingBiquant image analysis.

10. EXAMPLE COX-2 Expression in Amyotrophic Lateral Sclerosis

In situ hybridization has demonstrated increased COX-2 mRNA in theanterior horn cells of spinal cords of patients suffering fromamyotrophic lateral sclerosis (“SACS”); FIG. 16.

11. EXAMPLE Transient Expression of COX-2 In Neuronal Cells PotentiatesAβ Mediated Impairment of Redox Activity

The role of COX-2 in neuronal death and/or survival was studied incultured human SH-SY5Y neuronal cells. Cells were transientlytransfected with a mammalian expression vector (pRc/CMV/hCOX,Invitrogen) containing either a full length human (h) COX-2 cDNA(pRc/CMV/hCOX-2), or a bacterial chloramphenicol acetyltransferase (CAT)gene (pRc/CMV2/CAT). Following Aβ25-35 treatment, impairment of redoxactivity as assessed by3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetraxolium bromide (MTT)-assay(FIG. 19). Overexpression of hCOX2 in SH-SY5Y cells transfected withpRc/CMV/hCOX-2 was confirmed immunocytochemically in a parallel tissueculture chamber slide using an α-COX-2 antibody as previously described.In control cells transfected with pRc/CMV2/CAT, the same α-COX antibodygenerated minimal immunocytochemical signal. We found that transientoverexpression of hCOX-2 in SH-SY5Y neuronal cells potentiated Aβ25-35(25 μM, 48 hrs treatment) mediated impairment of redox activity whencompared to SH-SY5Y cultures transfected with control vector (±±p<0.01vs Aβ/control vector; *p<0.001 vs CTL. These data are consistent withevidence showing induction of COX-2 mRNA coincidentally with impairmentof redox activity in SH-SY5Y neuronal cells (FIG. 11).

12. EXAMPLE Experiments Using COX-2 Transgenic Mice

Based on the evidence that overexpression of COX-2 in neuronal cells maypotentiate Aβ-mediated redox impairment and the observation thatneuronal COX-2 is elevated in AD brain and in experimentalneurodegeneration, we generated a transgenic mouse model with neuronaloverexpression of human (h) COX-2. To obtain overexpression of hCOX-2 inneurons, a hybrid gene was prepared in which the expression of a cDNAsequence containing the entire hCOX-2 coding region is regulated by therat neuron-specific enolase (NSE) promoter.

We obtained four lines with high expression of hCOX-2 mRNA expression.In one of them (NHC32), COX-2 mRNA was assessed by in situ hybridizationanalysis (FIG. 20A). We found that in NHC32 mouse line, has high levelsof hCOX-2 mRNA in the hippocampal formation, cerebral cortex and inother neuronal layers (FIG. 20B). No white matter level of HCOX-2 mRNAwere found using combined immunocytochemistry for NSE and hCOX-2 in situhybridization on the same tissue section, we confirmed the selectivityof hCOX-2 transgene expression to neuronal cells.

It has further been shown, using neuronal cultures derived from thetransgenic mice, that hCOX-2 overexpression potentiated Aβ mediatedresponse. As depicted in FIG. 21, studies showed that primary neuronalcultures derived from transgenic mice with neuronal COX-2 overexpression(NHC32, n=3), are most susceptible to aggregated Aβ25-35 peptidesmediated impairment of redox activity (MTT assay, Aβ25-35 25 μM for 48hrs), when compared to equally treated neuronal cultures derived fromwild type/control littermates (n=4). Moreover, when examinedmorphologically, Aβ25-35 treated neurons derived from transgenic hCOX-2mice revealed an intensified regression of neuronal processes (B, inset)when compared to wild type control Aβ25-35 treated neuronal cultures (A,inset).

13. EXAMPLE Nimesulide Protects B12 Neuronal Cells Against GlutamateToxicity

We examined the role of nimesulide, indomethacin and NS398 (anotherCOX-2 preferential inhibitor) on glutamate mediated toxicity. In thisstudy we used B12 neuronal cells; toxicity was assessed by amountincrease of lactate dehydrogenase LDH levels in the conditioned mediumafter treatment. LDH is an index of cell toxicity and its elevation isgenerally accepted as marker of neurotoxicity. As shown in FIGS. 22-23,we found that nimesulide protected against glutamate toxicity (10 mM) at10−6, 10−9 and 10-12 M with pretreatment (24 hrs before) and cotreatment(with glutamate for 24 hrs). Indomethacin, protected at 10−6 M and lostits protective effect at 10−9 and 10-12 M. Marginal protection ofglutamate mediated toxicity by indomethacin was observed at 10−9 M(pretreatment 24 hrs). No protection was observed with NS398pretreatment. In the pretreatment condition drugs remained in the mediaduring glutamate treatment.

Various publications are cited herein, the contents of which are herebyincorporated in their entireties.

                   #             SEQUENCE LISTING<160> NUMBER OF SEQ ID NOS: 1 <210> SEQ ID NO 1 <211> LENGTH: 16<212> TYPE: PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: synthetic peptide <400> SEQUENCE: 1Cys Asn Ala Ser Ala Ser His Ser Arg Leu As #p Asp Ile Asn Pro Thr 1               5   #                10   #                15

What is claimed is:
 1. A transgenic mouse whose genome comprises a geneencoding human cyclooxygenase-2 under the control of a neuron-specificpromoter, wherein neuronal cells of the mouse express humancyclooxygenase-2 mRNA, wherein the level of human cyclooxygenase-2 mRNAin the hippocampus of the mouse is increased relative to the level ofhuman cyclooxygenase-2 mRNA in cerebral white matter, and whereinneuronal cell cultures derived from said mouse are more susceptible toaggregated Aβ25-35 peptide-mediated impairment of redox activityrelative to those derived from a mouse non-transgenic for the humancyclooxygenase-2 gene.
 2. A neuronal cell culture prepared from thetransgenic mouse of claim 1 wherein neuronal cells of the cultureexpress human cyclooxygenase-2 mRNA at levels sufficient to increasetheir susceptibility to Aβ25-35 peptide-mediated impairment of redoxactivity relative to neuronal cultures derived from a mousenon-transgenic for the human cyclooxygenase-2 gene.